Malting and brewing are multi-million dollar industries. The annual added-value on barley production alone was approximately US$3 billion, in the USA between 1983 and 1992. During the same period, the US brewing industry generated US$167 billion in total business activity. The economic benefits to be derived from increased productivity of the brewing industry worldwide are enormous. A major contributing factor to this increased productivity is the improvement of the input raw material, the barley crop, the quality of which is largely determined by its genetic profile.
The primary goal of barley improvement for use in the brewing industry is to expand the germplasm available to breeders, thereby making available elite cultivars which are higher yielding, disease resistant, and/or possess improved malting characteristics. Traditional plant breeding methods have made considerable progress toward these goals, however such processes are labour-intensive, imprecise and protracted, requiring several generations of genetic crosses to produce a substantially-improved genetic stock. The present invention is a significant advance in the improvement of malting characteristics of plant materials utilised in the brewing industry, in particular barley, wherein said invention provides a "master switch", controlling the expression of several malting genes, for example .alpha.-amylase and .beta.-glucanase, amongst others.
During the malting process, the barley grain is water-steeped for 1-2 days at 10-20.degree. C. to remove CO.sub.2, replace oxygen and dissipate heat. The steeping process induces germination of the seed, characterised by cell elongation and increased respiration in the embryo, stimulation of embryo secretions, protein biosynthesis and enzyme activation, and the initiation of endosperm hydration. Kernel moisture content increases from 10-15% (w/w) to 40-45% (w/w) as a result of steeping (botanically-defined germination initiates at approximately 30% moisture content). Following steeping, the grains are germinated in a controlled growth environment for 3-6 days, producing a "green malt". Losses may be incurred at this stage, from incomplete or variable germination of the seed and consequently, there are many benefits to be derived from producing a crop in which seed dormancy could be broken uniformly and completely during steeping, providing rapid and uniform germination. Furthermore, dormancy of barley seed should also be adequate to prevent pre-harvest sprouting and consequential crop losses. Accordingly, the present invention provides a means of regulating the expression of the .alpha.-amylase gene in aleurone cells of the seed. The present invention may be used to control the germination of a crop seed.
During malting, the major constituent of the cell wall of the starchy endosperm of barley, .beta.-glucan, is broken down by the enzyme (1-3, 1-4)-.beta.-glucanase, referred to hereinafter as .beta.-glucanase, which is secreted from the aleurone cells (Fincher, 1989). The efficient degradation of .beta.-glucan polymers is important to the brewing process, since the presence of high molecular weight .beta.-glucans increases the viscosity of the "mash", thereby slowing later filtration steps. Incomplete hydrolysis of .beta.-glucan molecules may even produce a cloudy precipitate in the fished product. The degree of hydrolysis of .beta.-glucan is a function of the level of .beta.-glucanase enzyme produced by the aleurone and the proportion of enzyme activity remaining following higher temperature incubations of kilning and mashing (see below). There is a clear need in the malting industry for the production of barley lines with increased .beta.-glucanase activity, to facilitate the malting process.
The green malt is dried in kilns to reduce kernel moisture to 3-5%. Malt components are subsequently converted into a fermentable substrate which includes sugars, amino acids, nucleic acids, vitamins and minerals. The malt is placed in warm water and taken through a series of controlled temperature rises and holds from 40.degree. C. to 75.degree. C., in order to gelatinise and solubilise seed starch reserves and to solubilise carbohydrate-degrading enzymes. The production of fermentable sugars for example maltose and glucose, requires the hydrolytic enzymes .alpha.-amylase, .beta.-amylase, .alpha.-glucosidase and limit dextrinase. Of these enzymes, .alpha.-amylase, .alpha.-glucosidase and limit dextrinase are known to be secreted from the aleurone (Fincher, 1989).
Fermentable sugars are produced by .alpha.-amylase enzyme activity at 70-75.degree. C. The survival of these hydrolytic enzymes, in particular .alpha.-amylase, during and after kilning, is critical to the brewing process and to the flavour and colour of the malt and the alcohol content of the finished product. The proportion of these enzymes remaining after kilning is directly proportional to the amount of enzyme in the germinating seed after dormancy is broken. Using technology available until the present invention, the efficiency of this process was improved by the addition of a microbial amyloglucosidase supplement to the mash, in particular in the production of some low calorie beers.
Although the development of a barley crop with improved malting characteristics, in particular possessing increased aleurone .alpha.-amylase, .alpha.-glucosidase, limit dextrinase, .beta.-glucanase, endoxylanase and protease activities, is highly desirable, traditional breeding technologies have not addressed the problem, in part because reliable methods for screening large numbers of plants carrying these trials have not been developed.
The level of aleurone secretory enzymes, in particular .alpha.-amylase, .beta.-glucanase, etc., may be increased by the application of the plant hormone, gibberellin acid (GA), in particular GA.sub.3 (Paleg, 1960; Varner, 1964; Yomo, 1960). Following addition of GA, there is a rapid rise in .alpha.-amylase gene expression in isolated barley aleurone layers and this effect is inhibited by ascisic acid (Jacobsen et al., 1995).
Although there are now considerable data about the site of GA perception in aleurone cells, the GA receptor has not yet been identified. Evidence from experiments using GA.sub.4 covalently bound to Sepharose beads and anti-idiotype antibodies suggests that GA is perceived on the plasma membrane in oat aleurone protoplasts (Hooley et al., 1991; Hooley et al., 1992). This is supported by recent work which shows micro injection of GA.sub.3 into isolated barley aleurone protoplasts failed to induce .alpha.-amylase synthesis and secretion (Gilroy and Jones, 1994). However, when GA.sub.3 was applied external to the medium, the protoplasts responded by increasing .alpha.-amylase gene expression, indicating that the site of perception is on the external face of the plasma membrane. Little is known of the molecular events downstream of the GA receptor which transmit the GA signal through the cytoplasm and ultimately trigger expression of genes encoding .alpha.-amylase and other hydrolytic enzymes (Bush and Jones, 1990; Gilroy and Jones 1992).
Functional analysis of barley high-pI .alpha.-amylase promoters have identified a gibberellin response complex (GARC) consisting of the pyrimidine, TAACAAA and TATCCAC boxes, which mediate the GA response (Skriver et al., 1991; Gubler and Jacobsen, 1992; Gubler et al., 1995). There is also evidence that the action of abscisic acid is mediated via the same complex. Analyses of a barley low-pI amylase promoter, have shown that GA probably also acts through similar cis-acting elements, but additional cis-acting elements upstream of the pyrimidine box are also important (Lanahan et al., 1992). DNA sequences that bind nuclear proteins in vitro have been identified in cereal .alpha.-amylase promoters using DNase 1 foot printing and gel mobility shift assays (Ou-Lee et al., 1988; Rushton et al., 1992; Sutliff et al., 1993; Goldman et al., 1994). Two recent studies have shown that GARC sequences in wheat and barley .alpha.-amylase promoters can act as binding sites for nuclear transcription factors which regulate gene expression. Sutliff et al. (1993) characterized nuclear factors from barley aleurone layers which bound in vitro, to sequences from a barley low-pI .alpha.-amylase gene. A GA-dependent binding factor was shown to bind specifically to sequences which coincide with the TAACAGA and TATCCAT boxes and proximal sequences. It is not yet clear whether this binding factor contains a single nuclear protein which binds to both elements or whether it consists of two or more proteins with different binding specificities. Rushton et al. (1992) demonstrated that nuclear factors from GA-treated oat protoplasts bound specifically to the box 2 and the pyrimidine and TAACAGA elements in a low-pI wheat .alpha.-amylase promoter. The function of these proteins in regulating .alpha.-amylase gene expression has not been determined. Furthermore, the demonstration of a DNA-protein interaction does not provide significant direction to enable a person normally skilled in the art to isolate the DNA-binding protein or a gene encoding said protein, or to determine the role of said protein in regulating the expression of genes encoding malting enzymes, for example .alpha.-amylase and .beta.-glucanase, amongst others.